Immunoassay test kit for measuring bone resorption

ABSTRACT

An immunoassay test kit including an immunological binding partner that binds to lysyl pyridinoline, for analyzing a body fluid sample for bone resorption in vivo.

This invention was made with government support under one or more ofgrants AM 26489, AR 37318, AM 30774, and AR 36794 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

This is a continuation of prior application Ser. No. 09/209,864, filedDec. 11, 1998, which is a continuation of application Ser. No.09/047,268, filed Mar. 24, 1998, which is a continuation of applicationSer. No. 08/771,452, filed Dec. 20, 1996, now abandoned, which is acontinuation of application Ser. No. 08/497,731, filed Jun. 21, 1995,now U.S. Pat. No. 5,607,862, which is a continuation of application Ser.No. 08/195,323, filed Feb. 10, 1994, now abandoned, which is acontinuation of application Ser. No. 07/840,574, filed Feb. 24, 1992,now abandoned, which is a continuation of application Ser. No.07/592,511, filed Oct. 3, 1990, now abandoned, which is a continuationof application Ser. No. 07/118,234, filed Nov. 6, 1987, now U.S. Pat.No. 4,973,666, priority from the filing dates of which is hereby claimedunder 35 U.S.C. § 120.

TECHNICAL FIELD

This invention relates to a method for assaying bone resorption rates.More specifically, it relates to a method for quantitating specificurinary cross-linking amino acids, and peptide fragments that containthose amino acids derived from degraded bone collagen.

BACKGROUND OF THE INVENTION

Osteoporosis is the most common bone disease in man. Primaryosteoporosis, with increased susceptibility to fractures, results from aprogressive net loss of skeletal bone mass. It is estimated to affect15-20 million individuals in the United States. Its basis is anage-dependent imbalance in bone remodelling, i.e., in the rates ofsynthesis and degradation of bone tissue. About 1.2 millionosteoporosis-related fractures occur in the elderly each year includingabout 538,000 compression fractures of the spine, about 227,000 hipfractures and a substantial number of early fractured peripheral bones.Twelve to 20% of the hip fractures are fatal because they cause severetrauma and bleeding, and half of the surviving patients require nursinghome care. Total costs from osteoporosis-related injuries now amount toat least $7 billion annually (Barnes, O.M., Science, 236, 914 (1987)).Osteoporosis is most common in postmenopausal women who, on average,lose 15% of their bone mass in the 10 years after menopause. Thisdisease also occurs in men as they get older and in young amenorrheicwomen athletes. Despite the major, and growing, social and economicconsequences of osteoporosis, no method is available for measuring boneresorption rates in patients or normal subjects. A major difficulty inmonitoring the disease is the lack of a specific assay for measuringbone resorption rates.

Methods for assessing bone mass often rely on measuring whole-bodycalcium by neutron activation analysis or mineral mass in a given boneby photon absorption techniques. These measurements can give onlylong-term impressions of whether bone mass is decreasing. Measuringcalcium balances by comparing intake with output is tedious, unreliableand can only indirectly appraise whether bone mineral is being lost overthe long term. Other methods currently available for assessing decreasedbone mass and altered bone metabolism include quantitative scanningradiometry at selected bone locations (wrist, calcaneus, etc.) andhistomorphometry of iliac crest biopsies. The former provides a crudemeasure of the bone mineral content at a specific site In a single bone.Histomorphometry gives a semi-quantitative assessment of the balancebetween newly deposited bone seams and resorbing surfaces.

A urinary assay for the whole-body output of degraded bone in 24 hourswould be much more useful. Mineral studies (e.g., calcium balance)cannot do this reliably or easily. Since bone resorption involvesdegradation of the mineral and the organic matrix, a specificbiochemical marker for newly degraded bone products in body fluids wouldbe the ideal index. Several potential organic indices have been tested.For example, hydroxyproline, an amino acid largely restricted tocollagen, and the principal structural protein in bone and all otherconnective tissues, is excreted in urine. Its excretion rate is known tobe increased in certain conditions, notably Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased. For thisreason, urinary hydroxyproline has been used extensively as an aminoacid marker for collagen degradation. Singer, F. R., et al. (1978) In:Metabolic Bone Disease, Vol. II (eds. Avioli, L. V. and Krane, S. M.)pp. 489-575, Academic Press, New York.

Goverde (U.S. Pat. No. 3,600,132) discloses a process for determinationof hydroxyproline in body fluids such as serum, urine, lumbar fluid andother intercellular fluids in order to monitor deviations in collagenmetabolism. In particular, this inventor notes that in pathologicconditions such as Paget's disease, Marfan's syndrome, osteogenesisimperfecta, neoplastic growth in collagen tissues and in various formsof dwarfism, increased collagen anabolism or catabolism as measured byhydroxyproline content in biological fluids can be determined. Thisinventor measures hydroxyproline by oxidizing it to a pyrrole compoundwith hydrogen peroxide and N-chloro-p-toluenesulphonamide followed bycolorimetric determination in p-dimethyl-amino-benzaldehyde.

In the case of Paget's disease, the increased urinary hydroxyprolineprobably comes largely from bone degradation, hydroxyproline, however,generally cannot be used as a specific index. Much of the hydroxyprolinein urine may come from new collagen synthesis (considerable amounts ofthe newly made protein are degraded and excreted without ever becomingincorporated into tissue fabric), and from turnover of certain bloodproteins as well as other proteins that contain hydroxyproline.Furthermore, about 80% of the free hydroxyproline derived from proteindegradation is metabolized in the liver and never appears in the urine.Kiviriko, K. I. (1970) Int. Rev. Connect. Tissue Res. 5, 93, and Weiss,P. H. and Klein, L. (1969) J. Clin. Invest. 48, 1.

Hydroxylysine and Its glycoside derivatives, both peculiar tocollagenous proteins, have been considered to be more accurate thanhydroxyproline as markers of collagen degradation. However, for the samereasons described above for hydroxyproline, hydroxylysine and itsglycosides are probably equally non-specific markers of bone resorption.Krane, S. M. and Simon, L. S. (1981) Develop. Biochem. 22, 185.

In addition to amino acids unique to collagen, various non-collagenousproteins of bone matrix such as osteocalcin, or their breakdownproducts, have formed the basis of immunoassays aimed at measuring bonemetabolism. Price, P. A. et al. (1980) J. Clin. Invest. 66, 878, andGundberg, C. M. et al. (1984) Meth. Enzymol. 107, 516. However, it isnow clear that bone-derived non-collagenous proteins, though potentiallya useful index of bone metabolic activity are unlikely, on their own, toprovide quantitative measures of bone resorption. The concentration inserum of osteocalcin, for example, fluctuates quite widely both normallyand in metabolic bone disease. Its concentration is elevated in statesof high skeletal turnover but it is unclear whether this results fromincreased synthesis or degradation of bone. Krane, S. M., et al. (1981)Develop. Biochem. 22, 185, Price, P. A. et al. (1980) J. Clin. Invest.66, 878, and Gundberg, C. M. et al. (1984) Meth. Enzymol. 107, 516.

Collagen Cross-Linking

The polymers of most genetic types of vertebrate collagen require theformation of aldehyde-mediated cross-links for normal function. Collagenaldehydes are derived from a few specific lysine or hydroxylysineside-chains by the action of lysyl oxidase. Various di-, tri- andtetrafunctional cross-linking amino acids are formed by the spontaneousintra- and intermolecular reactions of these aldehydes within the newlyformed collagen polymers; the type of cross-linking residue variesspecifically with tissue type (see Eyre, D. R. et al. (1984) Ann. Rev.Biochem. 53: 717-748). Two basic pathways of cross-linking can bedifferentiated for the banded (67 nm repeat) fibrillar collagens, onebased on lysine aldehydes, the other on hydroxylysine aldehydes. Thelysine aldehyde pathway dominates in adult skin, cornea, sclera, and rattail tendon and also frequently occurs in other soft connective tissues.The hydroxylysine aldehyde pathway dominates in bone, cartilage,ligament, most tendons and most internal connective tissues of the body,Eyre, D. R. et al. (1974) vida supra. The operating pathway is governedby whether lysine residues are hydroxylated in the telopeptide siteswhere aldehyde residues will later be formed by lysyl oxidase (Barnes,M. J. et al. (1974) Biochem. J. 139, 461). The chemical structure(s) ofthe mature cross-linking amino acids on the lysine aldehyde pathway areunknown, but hydroxypyridinium residues have been identified as matureproducts on the hydroxylysine aldehyde route. On both pathways and inmost tissues the intermediate, borohydride-reducible cross-linkingresidues disappear as the newly formed collagen matures, suggesting thatthey are relatively short-lived intermediates (Bailey, A. J. et al.(1971) FEBS Lett. 16, 86). Exceptions are bone and dentin, where thereducible residues persist in appreciable concentration throughout life,in part apparently because the rapid mineralization of the newly madecollagen fibrils inhibits further spontaneous cross-linking interactions(Eyre, D. R. (1981) In: The Chemistry and Biology of MineralizedConnective Tissues (Veis, A. ed.) pp. 51-55, Elsevier, New York, andWalters, C. et al. (1983) Caic. Tiss. Intl. 35: 401-405).

Two chemical forms of 3-hydroxypyridinium cross-link have beenidentified (Formula I and II). Both compounds are naturally fluorescent,with the same characteristic excitation and emission spectra (Fujimoto,D. et al. (1977) Biochem. Biophys. Res. Commun. 76, 1124, and Eyre, D.R. (1981) Develop. Biochem. 22, 50). These amino acids can be resolvedand assayed directly in tissue hydrolysates with good sensitivity usingreverse phase HPLC and fluorescence detection. Eyre, D. R. et al. (1984)Analyt. Biochem. 137: 380-388. ##STR1##

In growing animals it has been reported that these mature cross-linksmay be concentrated more in an unmineralized fraction of bone collagenthan in the mineralized collagen (Banes, A. J., et al. (1983) Biochem.Biophys. Res. Commun. 113, 1975). However, other studies on young bovineor adult human bone do not support this concept, Eyre, D. R. (1985) In:The Chemistry and Biology of Mineralized Tissues (Butler, W. T. ed.) p105, Ebsco Media Inc., Birmingham, Ala.

The presence of collagen hydroxypyridinium cross-links in human urinewas first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. andBoucek, R. J. (1981) Biochem J. 197: 759-762) using lengthy isolationprocedures for peptides and conventional amino acid analysis. At thattime, they were aware only of the HP form of the cross-link. Robins(Robins, S. P. (1982) Biochem J. 207: 617-820) has reported anenzyme-linked immunoassay to measure HP in urine, having raisedpolyclonal antibodies to the free amino acid conjugated to bovine serumalbumin. This assay is intended to provide an index for monitoringincreased joint destruction that occurs with arthritic diseases and isbased, according to Robins, on the finding that pyridinoline is muchmore prevalent in cartilage than in bone collagen. In more recent workinvolving enzyme-linked immunoassay, Robins reports that lysylpyridinoline is unreactive toward antiserum to pyridinoline covalentlylinked to bovine serum albumin (Robins et al. (1986) Ann. Rheum.Diseases 45, 969-973). Robins' urinary index for cartilage destructionis based on the discovery that hydroxylysyl pyridinoline, derivedprimarily from cartilage, is found in urine at concentrationsproportional to the rate of joint cartilage resorption. In principal,this index could be used to measure whole body cartilage loss, however,no information on bone resorption would be available.

A need therefore exists for a method that allows the measurement ofwhole-body bone resorption rates in humans. The most useful such methodwould be one that could be applied to body fluids, especially urine. Themethod should be sensitive, i.e., quantifiable down to 1 picomole andrapidly measure 24-hour bone resorption rates so that the progress ofvarious therapies (e.g., estrogen) can be assessed.

SUMMARY OF THE INVENTION

A method for determining the absolute rate of bone resorption comprisingquantitating the concentration of peptide fragments having3-hydroxypyridinium cross-links derived from bone collagen resorption ina body fluid. The quantitating steps consists of contacting the bodyfluid with an immunological binding partner specific to a peptidefragment having 3-hydroxypyridinium cross-links derived from bonecollagen resorption. In one embodiment of the invention, the body fluidis optionally purified prior to the contacting step. This purificationstep is selected from a number of standard procedures, includingcartridge adsorption and elution, molecular sieve chromatography,dialysis, ion exchange, alumina chromatography, hydroxyapatitechromatography, and combinations thereof.

The invention also encompasses other methods for quantitating theconcentration of peptide fragments having 3-hydroxypyridiniumcross-links in a body fluid. These methods include electrochemicaltitration, natural fluorescence spectroscopy, and ultravioletabsorbance. Electrochemical titration may be conducted directly upon abody fluid without further purification. However, when this is notpossible due to excessive quantities of contaminating substances, thebody fluid is first purified prior to the electrochemical titrationstep. Suitable methods for purification prior to electrochemicaldetection include dialysis, ion exchange chromatography, aluminachromatography, molecular sieve chromatography, hydroxyapatitechromatography and ion exchange absorption and elution.

Fluorometric measurement of a body fluid containing 3-hydroxypyridiniumcross-links is an alternative way of quantitating bone resorption. Thefluorometric assay can be conducted directly on a body fluid withoutfurther purification. However, for certain body fluids, particularlyurine, it is preferred that purification of the body fluid be conductedprior to fluorometric assay. This purification step consists ofdialyzing an aliquot of urine against an aqueous solution therebyproducing partially purified peptide fragments retained within thenondiffusate. The nondiffusate is then lyophylized, dissolved in an ionpairing solution and absorbed onto an affinity chromatography column.The chromatography column is washed with a volume of ion pairingsolution and, thereafter, the peptide fragments are eluted from thecolumn with an eluting solution. These purified peptide fragments arethen hydrolyzed and the hydrolyzate is resolved chromatographically.Chromatographic resolution is conducted by either high-performanceliquid chromatography or microbore high performance liquidchromatography.

The invention includes a peptide fragment derived from bone collagensubstantially free from other human peptides obtained from a body fluid.The peptide fragment may contain 3-hydroxypyridinium cross-links, inparticular, lysyl pyridinoline cross-links and hydroxylysyl pyridinolinecross-links.

A specific peptide fragment having a 3-hydroxpyridinium cross-linkderived from the aminoterminal telopeptide domain of bone type Icollagen has the following amino acid sequence. ##STR2## is hydroxylysylpyridinoline or lysyl pyridinoline and, Gln is glutamine or whollycyclized pyrrolidone carboxylic acid.

The invention also encompasses a peptide fragment containing3-hydroxypyridinium cross-links derived from the carboxyterminaltelopeptide domain of bone type I collagen. These carboxyterminaltelopeptide sequences are cross-linked with either lysyl pyridinoline orhydroxylysyl pyridinoline. An example of such a peptide sequence isrepresented by the formula: ##STR3## is hydroxylysyl or lysylpyridinoline.

The invention includes a fused cell hydrid which produces monoclonalanti-bodies specific for the peptide fragment derived from bone collagenhaving 3-hydroxypyridinium cross-links. The invention also includesmonoclonal antibodies produced by the fused cell hybrid including thoseantibodies coupled to a detectable marker. Examples of detectablemarkers include enzymes, chromophores, fluorophores, coenzymes, enzymeinhibitors, chemiluminescent materials, paramagnetic metals, spin labelsand radio nucleotides. The invention includes a test kit useful forquantitating the amount of peptide fragments having 3-hydroxypyridiniumcross-links derived from bone collagen resorption found in a body fluidcomprising the monoclonal antibody specific for peptide fragmentsderived from bone collagen and containing 3-hydroxypyridiniumcross-links. The monoclonal antibodies of this test kit may be coupledto the detectable markers described above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is based on the discovery that both lysyl pyridinoline(LP) and hydroxylysyl pyridinoline (HP) peptide fragments derived fromreabsorbed bone collagen are excreted in the urine without beingmetabolized. The invention is also based on the discovery that no otherconnective tissues contain significant levels of LP and that the ratioof HP to LP in mature bone collagen remains relatively constant over aperson's lifetime.

FIG. 1 compares the concentration of HP and LP in both cortical andcancellous human bone with age. It is observed that the concentration ofHP plus LP cross-links in bone collagen reaches a maximum by age 10 to15 years and remains reasonably constant throughout adult life.Furthermore, the ratio of HP to LP, shown in FIG. 2, shows little changethroughout life, remaining constant at about 3.5 to 1. These baselinedata demonstrate that the 3-hydroxypyridinium cross-links in bonecollagen remains relatively constant and therefore that body fluidsderived from bone collagen degradation will contain 3-hydroxypyridiniumcross-linked peptide fragments at concentrations proportional to theabsolute rate of bone resorption.

Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen,the method for determining the absolute rate of bone resorption, in itssimplest form, is based on quantitating the concentration of peptidefragments containing 3-hydroxypyridinium cross-links and preferablylysyl pyridinoline (LP) cross-links in a body fluid. As used in thisdescription and in the appended claims, by quantitating is meantmeasuring by any suitable means, including but not limited tospectrophotometric, gravimetric, volumetric, coulometric, immunometric,potentiometric, or amperometric means the concentration of peptidefragments containing 3-hydroxypyridinium cross-links in an aliquot of abody fluid. Suitable body fluids include urine, serum, and synovialfluid. The preferred body fluid is urine.

Since the concentration of urinary peptides will decrease as the volumeof urine increases, it is further preferred that when urine is the bodyfluid selected, the aliquot assayed be from a combined pool of urinecollected over a fixed period of time, for example, 24 hours. In thisway, the absolute rate of bone resorption is calculated for a 24 hourperiod. Alternatively, urinary peptides may be measured as a ratiorelative to a marker substance found in urine such as creatinine. Inthis way the urinary index of bone resorption would remain independentof urine volume.

In one embodiment of the present invention, monoclonal or polyclonalanti-bodies are produced which are specific to the peptide fragmentscontaining lysyl pyridinoline cross-links found in urine. Peptidefragments may be isolated from the urine of any patient, however, it ispreferred that these peptides are isolated from patients with Paget'sdisease, due to the high concentration of peptide fragments found inthese patients.

ISOLATION OF URINARY PEPTTIDES

Urine from patients with active Paget's disease is dialyzed in reducedporosity dialysis tubing (>3,500 Spectropore) at 4° C. for 48 h toremove bulk solutes. Under these conditions the peptides of interest arelargely retained. The freeze-dried non-diffusate is then eluted (200 mgaliquots) from a column (90 cm×2.5 cm) of Bio-Gel P2 (200-400 mesh) in10% acetic acid at room temperature. A region of effluent that combinesthe cross-linked peptides is defined by measuring the fluorescence ofcollected fractions at 297 nm excitation/395 nm emission, and this poolis freeze-dried. Further resolution of this material is obtained on acolumn of Bio-Gel P-4 (200-400 mesh, 90 cm×2.5 cm) eluted in 10% aceticacid. Two contiguous fraction pools are defined by monitoring thefluorescence of the eluant above. The earlier fraction is enriched inpeptide fragments having two amino acid sequences that derive from thecarboxyterminal telopeptide domain of the αI(I) chain of bone type Icollagen linked to a third sequence derived from the triple-helical bodyof bone type I collagen. These three peptide sequences are cross-linkedwith 3-hydroxypyridinium. The overlapping later fraction is enriched inpeptide fragments having an amino acid sequence that derives from theaminoterminal telopeptide domain of bone type I collagen linked througha 3-hydroxypyridinium cross-links. Individual peptides are then resolvedfrom each of the two fractions obtained above by ion-exchange HPLC on aTSK DEAE-5-PW column (Bio Rad 7.5 cm×7.5 mm) eluting with a gradient ofNaCl (0-0.2M) in 0.02M Tris-HCl, pH 7.5 containing 10% (v/v)acetonitrile. The aminoterminal telopeptide-based and carboxyterminaltelopeptide-based cross-linked peptides elute in a series of 3-4 peaksof fluorescence between 0.08M and 0.15M NaCl. The carboxyterminaltelopeptide-based cross-linked peptides elute first as a series offluorescent peaks, and the major and minor aminoterminaltelopeptide-based cross-linked peptides elute towards the end of thegradient as characteristic peaks. Each of these is collected,freeze-dried and chromatographed on a C-18 reverse phase HPLC column(Vydac 218TP54, 25 cm×4.6 mm) eluted with a gradient (0-10%) ofacetonitrile: n-propanol (3:1 v/v) in 0.01M trifluoroacetic acid. About100-500 μg of Individual peptide fragments containing3-hydroxypyridinium cross-links can be isolated by this procedure from asingle 24 h collection of Paget's urine. Amino acid compositions of themajor isolated peptides confirmed purity and molecular sizes by thewhole number stoichibmetry of recovered amino acids. Aminoterminalsequence analysis by Edman degradation confirmed the basic corestructures suspected from the sequences of the known cross-linking sitesin type I collagen and from the matching amino acid compositions. Theaminoterminal telopeptide sequence of the α2(I) chain was blocked fromsequencing analysis due presumably to the known cyclization of theaminoterminal glutamine to pyrrolidone carboxylic acid. A typicalelution profile of aminoterminal telopeptides obtained by the aboveprocedure is shown in FIG. 3a. The major peptide fragment obtained hasan amino acid composition: (Asx)₂ (Glx)₂ (Gly)₅ Val-Tyr-Ser-Thr, whereAsx is the amino acid Asp or Asn and Glx is the amino acid Gln or Glu.The sequence of this peptide is represented by Formula III below.

The carboxyterminal telopeptide-based cross-linked peptides resolved byreverse phase HPLC as described above are shown in FIG. 3b. As can beseen from this figure, these peptides are further resolved into a seriesof carboxyterminal telopeptides each containing the 3-hydroxypyridiniumcross-links. The major peptide, shown in FIG. 3b, was analyzed asdescribed above and was found to have the amino acid composition: (Asp)₅(Glu)₄ (Gly)₁₀ (His)₂ (Arg)₂ (Hyp)₂ (Ala)₅. The sequence of this peptideis represented by formula IV below. It is believed that the othercarboxyterminal telopeptide-based cross-linked peptides appearing asminor peaks in FIG. 3b represent additions and deletions of amino acidsto the structure shown in Formula IV. Any of the peptides containedwithin these minor peaks are suitable for use as immunogens as describedbelow. ##STR4## where ##STR5## represents the HP or LP cross-linkingamino acids, and

Gln represents glutamine or a wholly cyclized pyrrolidone carboxylicacid.

Equivalents of the peptides represented by the above structures includethose cases where some variation in the urinary peptide structureaccrues. Examples of variation include amino acid additions to the N andC termini of Formulae III and IV as well as some terminal amino aciddeletions. Smaller peptide fragments of the molecule represented byFormula IV derived from bone readsorption are especially evident inurine. These are found in the minor peaks of the carboxytelopeptidefraction seen in FIG. 3b and can be identified by amino acid compositionand sequence analysis. Furthermore, both the Ser and Thr residues ofFormula III are occasionally conjugated to a small molecule. It isanticipated that antibodies produced to the haptens represented byFormulae III and IV will cross react with urinary peptides of slightlyvaried structure. In some situations it may be desirable to producepatient-specific antibodies to the urinary peptides derived from boneresorption. In these cases the same procedure described above isutilized to isolate urinary peptides whose structure may vary slightlyfrom that represented by Formulae III and IV.

IMMUNOLOGICAL PROCEDURE FOR INDEXING BONE RESORPTION

Immunological binding partners capable of specifically binding topeptide fragments derived from bone collagen obtained from aphysiological fluid can be prepared by methods well known in the art.The preferred method for isolating these peptide fragments is describedabove. By immunological binding partners as used herein is meantantibodies and antibody fragments.

Both monoclonal and polygonal antibodies specifically binding thepeptides represented by Formulae III and IV and their equivalents areprepared by methods known in the art. For example, Laboratory Techniquesin Biochemistry and in Molecular Biology, Campbell, A. M. (1986) Vol. 13Elsevier, herein incorporated by reference. It is possible to produceantibodies to the above peptides or their equivalents as isolated.However, because the molecular weights of these peptide fragments areless than 5,000, it is preferred that the hapten be conjugated to acarrier molecule. Suitable carrier molecules include, but are notlimited to, bovine serum albumin, ovalbumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). Preferred carriers are thyroglobulin and KLH.

It is well known in the art that the orientation of the hapten, as it isbound to the carrier protein, is of critical importance to thespecificity of the anti-serum. Furthermore, not all hapten-proteinconjugates are equally successful immunogens. The selection of aprotocol for binding the particular hapten to the carrier proteintherefore depends on the amino acid sequence of the urinary peptidefragments selected. For example, if the urinary peptide fragmentrepresented by Formula III is selected, a preferred protocol wouldinvolve coupling this hapten to keyhole limpet hemocyanin (KLH), orother suitable carrier, with carbodiimide. This would ensure that mostof the hapten would be conjugated through the Gly carboxyterminus,thereby presenting the preferred epitope, namely Tyr and3-hydroxypyridinium cross-link, to the primed vertebrate antibodyproducing cells (e.g., B-lymphocytes).

Other urinary peptide fragments, depending on the source, may requiredifferent binding protocols. Accordingly, a number of binding agents maybe suitably employed. These include, but are not limited to, carboduimides, glutar-aldehyde, mixed anhydrides, as well as bothhomobifunctional and heterobi-functional reagents (see for example thePierce 1986-87 catalog, Pierce Chemical Co., Rockford, Ill.). Preferredbinding agents include carbodiimides and heterobifunctional reagentssuch as m-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).

Methods for binding the hapten to the carrier molecule are known in theart. See for example Laboratory Techniques in Biochemistry and MolecularBiology, Chard, T. (1987) Vol. 6, Partz Elsevier, N.Y., hereinincorporated by reference.

Either monoclonal or polyclonal antibodies to the hapten-carriermolecule immunogen can be produced. However, it is preferred thatmonoclonal antibodies (MAb) be prepared. For this reason it is preferredthat immunization be carried out in the mouse. Immunization protocolsfor the mouse usually include an adjuvant. Examples of suitableprotocols are described by Chard, T. (1987) vida supra. Spleen cellsfrom the immunized mouse are harvested and homogenized and thereafterfused with cancer cells in the presence of polyethylene glycol toproduce a fused cell hybrid which produces monoclonal antibodiesspecific to peptide fragments derived from bone collagen. Examples ofsuch peptide fragments are represented by Formulae III and IV above.Suitable cancer cells include myeloma, hepatoma, carcinoma, and sarcomacells. Detailed descriptions of this procedure, including screeningprotocols, protocols for growing selected hybrid cells and harvestingmonoclonal antibodies produced by the selected hybrid cells are providedin Galfre, G. and Milstein, C. (1981) Meth. Enzymol. 73, 1. A preferredpreliminary screening protocol involves the use of peptide fragmentsderived from bone collagen resorption and containing 3-hydroxypyridiniumcross-links in a solid phase radioimmunoassay.

Immunological binding partners, especially monoclonal antibodies,produced by the above procedures, or equivalent procedures, are employedin various immunometric assays to quantitate the concentration ofpeptide fragments having 3-hydroxypyridinium cross-links derived frombone collagen resorption in body fluids. These immunometric assayscomprise a monoclonal antibody or antibody fragment coupled to adetectable marker. Examples of suitable detectable markers include butare not limited to: enzymes, coenzymes, enzyme inhibitors, chromophores,fluorophores, chemiluminescent materials, paramagnetic metals, spinlabels, and radionuclides. Examples of standard immunometric methodssuitable for indexing bone resorption include, but are not limited to,enzyme linked immunosorbent assay ELISA (Ingvall, E. (1981) Meth.Enzymol. 70), radio-immunoassay (RIA), and "sandwich" Immuno radiometricassay (IRMA). In its simplest form, these immunometric methods can beused to determine the absolute rate of bone resorption by simplycontacting a body fluid with the immunological binding partner specificto a peptide fragment having 3-hydroxypyridinium cross-links derivedfrom bone collagen resorption. It is preferred that the immunometricassays described above be conducted directly on untreated body fluids.Occasionally, however, contaminating substances may interfere with theassay necessitating partial purification of the body fluid. Partialpurification procedures include, but are not limited to, cartridgeadsorption and elution, molecular sieve chromatography, dialysis, ionexchange, alumina chromatography, hydroxyapatite chromatography andcombinations thereof.

Test kits, suitable for use in accordance with the present invention,contain monoclonal antibodies prepared as described above thatspecifically bind to peptide fragments having 3-hydroxypyridiniumcross-links derived from bone collagen resorption found in a body fluid.It is preferred that the monoclonal antibodies of this test kit becoupled to a detectable marker of the type described above.

ELECTROCHEMICAL PROCEDURE FOR INDEXING BONE RESORPTION

An alternative procedure for indexing bone resorption consists ofmeasuring a physical property of the peptide fragments having3-hydroxypyridinium cross-links. One such physical property suitable forindexing bone resorption relies upon electrochemical detection. Thismethod consists of injecting an aliquot of a body fluid, such as urine,into an electrochemical detector poised at a redox potential suitablefor detection of peptides containing the 3-hydroxypyridinium ring. The3-hydroxypyridinium ring, being a phenol, is subject to reversibleoxidation and therefore the electrochemical detector (e.g., Model 5100ACoulochem sold by esa 45 Wiggins Ave., Bedford, Mass.) is a highlydesirable instrument suitable for quantitating the concentration ofurinary peptides derived from bone adsorption. Two basic forms ofelectrochemical detector are currently commercially available:amperometric (e.g., BioAnalytical Systems) and coulometric (ESA, Inc.,Bedford, Mass. 01730). Both are suitable for use in accordance with thepresent invention, however, the latter system is inherently moresensitive and therefore preferred since complete oxidation or reductionof the analyzed molecule in the column effluent is achieved. Inaddition, screening or guard electrodes can be placed "upstream" fromthe analytical electrode to selectively oxidize or reduce interferingsubstances thereby greatly improving selectivity. Essentially, thevoltage of the analytical electrode is tuned to the redox potential ofthe sample molecule, and one or more pre-treatment cells are set todestroy interferents in the sample. In a preferred assay method, astandard current/voltage curve is established for standard peptidescontaining lysyl pyridinoline or hydroxylysyl pyridinoline in order todetermine the proper voltage to set for optimal sensitivity. Thisvoltage is then modified depending upon the body fluid, to minimizeinterference from contaminants and optimize sensitivity. Electrochemicaldetectors, and the optimum conditions for their use are known to thoseskilled in the art. Complex mixtures of body fluids can often bedirectly analyzed with the electrochemical detector withoutinterference. Accordingly, for most patients no pretreatment of the bodyfluid is necessary. In some cases however, interfering compounds mayreduce the reliability of the measurements. In such cases, pretreatmentof the body fluid (e.g ., urine) may be necessary.

Accordingly, in an alternative embodiment of the invention, a body fluidis first purified prior to electrochemically titrating the purifiedpeptide fragments. The purification step may be conducted in a varietyof ways including but not limited to; dialysis, ion exchangechromatography, alumina chromatography, hydroxyapatite chromatography,molecular sieve chromatography, or combinations thereof. In a preferredpurification protocol, a measured aliquot (25 ml) of a 24 hour urinesample is dialyzed in reduced porosity dialysis tubing to remove thebulk of contaminating fluorescent solutes. The non diffusate is thenlyophilized, redissolved in 1% heptafluorobutyric acid (HFBA), an ionpairing solution, and the peptides adsorbed on a Waters Sep-Pak C-18cartridge. This cartridge is then washed with 5 ml of 1% HFBA, and theneluted with 3 ml of 50% methanol in 1% HFBA.

Another preferred method of purification consists of adsorbing ameasured aliquot of urine onto an Ion-exchange adsorption filter andeluting the adsorption filter with a buffered eluting solution. Theeluate fractions containing peptide fragments having 3-hydroxypyridiniumcross-links are then collected to be assayed.

Still another preferred method of purification employs molecular sievechromatography. For example, an aliquot of urine is applied to a Bio-GelP2 or Sephadex G-20 column and the fraction eluting in the 1000-5000Dalton range is collected. It will be obvious to those skilled in theart that a combination of the above methods may be used to purify orpartially purify urine or other body fluids in order to isolate thepeptide fragments having 3-hydroxypyridinium cross-links. The purifiedor partially purified peptide fragments obtained by the above proceduresmay be subjected to additional purification procedures, furtherprocessed or assayed directly in the partially purified state.Additional purification procedures include resolving partially purifiedpeptide fragments employing high performance liquid chromatography(HPLC) or microbore HPLC when increased sensitivity is desired. Thesepeptides may then be quantitated by electrochemical titration. Apreferred electrochemical titration protocol consists of tuning theredox potential of the detecting cell of the electrochemical detector(Coulochem Model 5100A) for maximum signal with pure HP. The detector isthen used to monitor the effluant from a C-18 HPLC column used toresolve the partially purified urinary peptides.

FLUOROMETRIC PROCEDURE FOR INDEXING BONE RESORPTION

An alternative preferred method for quantitating the concentration ofpeptide fragments having 3-hydroxypyridinium cross-links is to measurethe characteristic natural fluorescence of these peptide fragments. Forthose body fluids containing few naturally occurring fluorescentmaterials other than the 3-hydroxypyridinium cross-links, fluorometricassay may be conducted directly without further purification of the bodyfluid. In this case, peptide fragments are resolved by HPLC and thenatural fluorescence of the HP and LP amino acid residues is measured at395 nm upon excitation at 297 nm, essentially as described by Eyre, D.R., et al., Analyl. Biochem. 137, 380 (1984), herein incorporated byreference.

It is preferred, in accordance with the present invention, that thefluorometric assay be conducted on urine. Urine, however, usuallycontains substantial amounts of naturally occurring fluorescentcontaminants that must be removed prior to conducting the fluorometricassay. Accordingly, urine samples are first partially purified asdescribed above for electrochemical detection. This partially purifiedurine sample can then be fluorometrically assayed as described above.Alternatively, the HP and LP cross-linked peptides in the partiallypurified urine samples or other body fluids can be hydrolyzed in 6M HClat about 108° C. for approximately 24 hours as described by Eyre, et al.(1984) vida supra. This process hydrolyzes the amino acids connected tothe lysine precursors of "tripeptide" HP and LP cross-links, producingthe free HP and LP amino acids represented by Formulae I and II. Thesesmall "tripeptides" are then resolved by the techniques described above,preferably by HPLC, and the natural fluorescence is measured (Ex 297 nm,Ex 390 nm).

Optionally, the body fluid (preferably urine) is passed directly througha C-18 reverse phase affinity cartridge after addingacetonitrile/methanol 5 to 10% V/V. The non-retentate is adjusted to0.05-0.10M with a cationic ion-pairing agent such as tetrabutyl ammoniumhydroxide and passed through a second C-18 reverse phase cartridge. Thewashed retentate, containing fluorescent peptides, from this secondcartridge is eluted with acetonitrile:water (or methanol:water), driedand fluorescent peptides are analyzed by reverse phase HPLC or microboreHPLC using an anionic ion-pairing agent such as 0.01M trifluoroaceticacid in the eluant.

FIG. 4A displays the elution profile resolved by reverse phase HPLC ofnatural fluorescence for a hydrolysate of peptide fragments from normalhuman urine. Measurement of the integrated area within the envelope of agiven component is used to determine the concentration of that componentwithin the sample. The ratio of HP:LP found in normal human urine andurine from patients having Paget's disease, FIG. 4B, are bothapproximately 4.5:1. This is slightly higher than the 4:1 ratio found inbone itself (Eyre, et al., 1984). The higher ratio found in urineindicates that a portion of the HP fraction in urine may come fromsources other than bone such as the diet, or other sources of co flagendegradation, i.e., cartilage catabolism. It is for this reason that itis preferred that LP which derives only from bone be used to provide anabsolute index of bone resorption. However, in the absence of excessivecartilage degradation such as in rheumatoid arthritis or in cases wherebone is rapidly being absorbed, HP or a combination of HP plus LP may beused as an index of bone resorption.

While the invention has been described in conjunction with preferredembodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein. It is therefore intended that theprotection granted by Letters Pat. hereon be limited only by theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. An immunoassay test kitfor analyzing a body fluid sample for the presence of analyte indicativeof bone resorption in vivo, comprising an immunological binding partnerthat binds to lysyl pyridinoline.
 2. The immunoassay test kit of claim1, wherein the immunological binding partner is selected from amongmonoclonal antibodies, polyclonal antibodies, and analyte-bindingfragments thereof.
 3. An enzyme linked immunosorbent assay (ELISA) testkit, comprising a monoclonal antibody that binds to lysyl pyridinoline.